Assay for motile facultative anaerobic pathogens

ABSTRACT

An approved assay for the determination for motile facultative anaerobic pathogens is described which involves the use of a growth-enhancing substance such as oxyrase enzyme in order to increase the growth rate of a target pathogen in a growth medium, to thereby shorten the time required to complete the assay. The assay of the invention is particularly suited for the determination of L. monocytogenes in food products such as meat and dairy goods, and provides an effective screening assay which can be completed in about 24-36 hours. Preferably, a product sample is first incubated in Fraser Broth containing oxyrase enzyme; if a darkened color results, a small portion of the liquid is then placed within a U-shaped culture tube provided with modified Oxford agar; the motility characteristics of the target pathogen are observed in the U-tube, which is indicative of the presence of L. monocytogenes.

This application is a continuation of application Ser. No. 07/936,529,filed Aug. 28, 1992, now abandoned which is a continuation of Ser. No.07/594,647, filed Oct. 9, 1990, now U.S. Pat. No. 5, 187,070.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is broadly concerned with an improved assay forthe determination of motile facultative anaerobic pathogens, andespecially L. monocytogenes, in samples such as meat. More particularly,it is concerned with such an assay which makes use of a substance suchas an oxygen-reactive enzyme (e.g., oxyrase enzyme) for enhancing thegrowth rate of a target pathogen in a selected growth medium, in orderto materially lessen the amount of time required for completing theassay. Assays for determining the presence of L. monocytogenes in meatsamples can be completed in times many hours shorter than previousstandard assays.

2. Description of the Prior Art

Listeria monocytogenes is a motile facultative anaerobic pathogen whichcan cause various diseases in man, including meningoencephalitis,low-grade septicemia, infectious mononucleosis-like syndrome, pneumonia,endocarditis, bacterial aortic aneurysm, localized abscesses, papular orpustular cutaneous lesions, conjunctivitis and urethritis. It is knownthat the pathogen can be transmitted from the eating of infected meat ormilk. Accordingly, because of the pernicious effects of this pathogen,and its increasing incidence in human foods, there has been asignificant increase in the concern expressed about L. monocytogenes andits effects on human health.

The accepted present-day assay for L. monocytogenes is described in"FSIS Method for the Isolation and Identification of Listeriamonocytogenes From Processed Meat and Poultry Products", LaboratoryCommunication No. 57, May 24, 1989, distributed by the USDA and others;this publication is incorporated by reference herein. Broadly speaking,the FSIS assay involves placing a sample of meat in UVM Broth followedby incubation at 30° C. for 24 hours. A 0.1 ml. aliquot of the incubatedmixture is then placed in 10 ml. of Fraser Broth and incubated at 30° C.or 24-48 hours. If the Fraser Broth darkens, the liquid is swabbed ontoa modified Oxford agar plate, which is then incubated at 35° C. for24-48 hours. The incubated plates are then examined for L. monocytogenescolonies exhibiting characteristic surrounding black zones resultingfrom hydrolyzed esculin. Suspected colonies of L. monocytogenes are thengently touched with an inoculation needle and are streaked for isolationonto a Horse Blood Overlay Agar plate. These plates are incubatedovernight at 35° C. Thereafter, the plates are examined under afluorescent lamp and translucent colonies are then further screened andsubjected to conventional confirming tests.

Generally speaking, the prior L. monocytogenes assay involves a totaltime of from 56-72 hours. This represents a real difficulty for the foodprocessor, in that food otherwise ready for shipment must be heldpending the completion of assay screening. Accordingly, there is a realneed in the art for an effective assay for L. monocytogenes (or othermotile facultative anaerobic pathogens) which can be successfullyperformed in a significantly shorter period of time.

SUMMARY OF THE INVENTION

The present invention overcomes the problems outlined above and providesan improved assay for motile facultative anaerobic pathogens which inthe case of L. monocytogenes can be completed in a total time of fromabout 24-36 hours. Broadly, the assay of the invention is designed todetermine the presence of target pathogens in a product sample (e.g.beef or other meats) capable of supporting the growth of such pathogens.The assay includes the steps of first incubating the sample in a growthmedium containing an agent such as esculin which will change the colorof the medium in the presence of the target pathogen. If such acharacteristic color change is observed, the motility characteristics ofthe pathogen for the color change is determined. The specificimprovement of the invention involves contacting the product sampleduring the incubation step with an effective amount of a substance whichenhances the growth rate of the target pathogen, this substance beingselected from the group consisting of growth-stimulating effectivereducing agents and oxygen-reactive enzymes. In particularly preferredforms, the growth medium is Fraser Broth, a known medium commercializedby, inter alia, Beeton Dickinson Microbiology Systems of Cockeysville,Md. A copy of a Becton Dickinson "Product Information Sheet" relative tothe Fraser Broth is incorporated by reference herein. While Fraser Brothis the preferred medium, those skilled in the art will appreciate thatother media can also be employed, so long as the media contains thedescribed color-change agent.

The most preferred growth-enhancing substance is oxyrase enzyme, knownto be an effective oxygen-reducing enzyme used to produce anaerobicconditions. The oxyrase enzyme is described in a technical bulletin"Properties of the Oxyrase Enzyme System Used to Isolate and CultivateAnaerobic Microorganisms", distributed by Oxyrase, Inc. of Ashland,Ohio. Moreover, the enzyme system is further described by Adler et al.in J. Bacteriology, August 1981, p.326-332; and in a manuscript of H. I.Adler dated Aug. 23, 1989 and scheduled to be published in 1990 andentitled "The Use of Microbial Membranes to Achieve Anaerobiosis." Allof these references are incorporated by reference herein.

The initial incubation step of the assay invention is carried out for aperiod of about 5-30 hours at a temperature of from about 25°-35° C. Ifthe target pathogen is present in the sample, and is incubated in anesculin-containing medium, the initial incubation will yield a darkbrown or blackened color, because of hydrolysis of the esculin. If nosuch darkening occurs, the sample is deemed free of the target pathogen.

In the next step of the preferred assay, a small aliquot (e.g., 1 ml.)of the darkened liquid resulting from the initial incubation is placedwithin one leg of a previously prepared U-shaped KIMAX culture tube. Thetube has a central bight section as well as a pair of spaced,upstanding, capped legs. A quantity of semisolid modified Oxford agar isplaced within the central section of the tube. and 1 ml. portions ofFraser Broth supplemented with a small amount of oxyrase enzyme areplaced atop the spaced ends of the agar. The modified Oxford agar is aknown product and is described in a Beeton Dickinson MicrobiologySystems "Product Information Sheet" for the modified agar. A copy ofthis publication is incorporated by reference herein. This Oxford agaris specially formulated as a selective medium for the isolation of L.monocytogenes from specimens containing a mixed flora.

L. monocytogenes exhibits a peculiar motility and, in the modifiedOxford agar, will gradually move through the agar and eventually darkenthe Fraser Broth sample in the opposite leg of the culture tube. Inparticular, after the culture tube is inoculated, incubation is carriedout at a temperature from about 30° to 40° C. for a period of about 3-15hours. If such darkening occurs, it is very, probable that the startingsample is contaminated with L. monocytogenes. However, in order toconfirm this, the sample is then assayed using conventional confirmingassays, such as those described in Bergey's Manual of SystematicBacteriology Vol. 2. Sec. 14. p. 1241.

Although a preferred assay in accordance with the invention involves thescreening determination of the presence of L. monocytogenes in beef. theinvention is not so limited. Thus, this target pathogen may be assayedfrom samples of other meats such as poultry (e.g. chicken), fish ordairy products. By the same token, other pathogens can also be assayedusing the principles of the invention. Such pathogens would include thegroup consisting of Listeria spp., Salmonella spp., E. coil, E. coli0157:H7, Campylobacter spp. Campylobacter jejuni, Yersinia spp.,Campylobacter jejuni, Yersinia spp., Yersinia enterocolitica, and allmembers of the Family Enterobacteiaceae.

Finally. although the use or an oxygen-reactive enzyme such as oxyraseis, preferred, useful results can be obtained by the use of reducingagents which are effective for creating anaerobic reaction conditionsand otherwise enhance the growth of target pathogens in a growth medium.Exemplary reducing agents include L-cysteine, HC1 and titanium (III)citrate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic process flow drawing illustrating the preferredassay for the determination of motile facultative anaerobic pathogens:

FIG. 2 is a graph illustrating the growth characteristics of the L.monocytogenes strain LM 101M in growth medium and in the presence ofoxyrase enzyme;

FIG. 3 is a graph illustrating the growth characteristics of the L.monocytogenes strain LM 103M in growth medium and in the presence ofoxyrase enzyme;

FIG. 4 is a graph illustrating the growth characteristics of the L.monocytogenes strain Scott A in growth medium and in the presence ofoxyrase enzyme;

FIG. 5 is a graph similar of that of FIGS. 2-4 but illustrating theeffect of oxyrase enzyme on S. typhimurium suspended in TSB medium;

FIG. 6 is a graph illustrating the effect of oxyrase enzyme on a strainof E. coli suspended in BHI medium;

FIG. 7 is a graph illustrating the growth characteristics of heatinjured L. monocytogenes contacted with oxyrase enzyme in a growthmedium; and

FIG. 8, is a graph illustrating the growth characteristics of L.monocytogenes in various growth media supplemented with esculin andiron.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1

The most preferred method of detecting L. monocytogenes in a samplecontaining from about 1-100 CFU/g is set forth below, with the FIG. 1illustration also being referred to as an aid to description. It is tobe understood that the following is given for illustrative purposes onlyand should not be considered as a limitation upon the overall scope ofthe invention.

First, 25 g. of the beef sample (cut into small pieces) is placed in aconventional anaerobic Stomacher bag 10 (see FIG. 1), along with 0.25units of oxyrase enzyme and 225 ml. of Fraser Broth. In particular, theBroth is first added to the bag, then the enzyme and finally the meatpieces. The bag 10 is scaled as at 12, and is placed into an incubatorat 30° C. The mixture is incubated at that temperature for a period offrom about 20-25 hours.

If L. monocytogenes is present in the sample, the liquid within the bagwill darken to a brownish-black color during the incubation period. Thisresults because of the fact that the target pathogen will metabolize theesculin contained in the broth, yielding the color change.

In the second step, a U-shaped culture tube 14 (such as the KIMAX tubedepicted in FIG. 1 having a U-shaped central section 16 and spaced,upstanding capped legs 18, 20) is prepared. In particular, a quantity ofthe semisolid Oxford modified agar 22 is placed within the section 16,and 1 ml. portions 24. 26 of Fraser Broth supplemented with 0.1 units ofoxyrase enzyme are placed atop the agar 22 as growth and confirmingliquids respectively. The ends of the tube 14 are then capped to ensureanaerobic conditions.

A 1 ml. sample of the darkened liquid within bag 10 and placed withintube 14, specifically within the liquid portion 24 shown in FIG. 1. Thetube is then placed within an incubator at 35° C. for a period of 5-11hours. L. monocytogenes exhibits a type of end-over-end tumblingmotility which enables it to travel through the liquid 24 and agar 22.Inasmuch as liquid 24 contains esculin, and the agar contains ferricammonium citrate and esculin, such characteristic motility may bereadily observed by a blackening of liquid 24, progressive blackening ofagar 22, and ultimately blackening of liquid 26.

At this point the assay has confirmed the presence of a motilefacultative anaerobic pathogen which is quite likely to be L.monocytogenes. However, in order to confirm the identity of the pathogenafter the foregoing screening assay, a sample of the starting meatproduct is then subjected to known confirming assays specific for L.monocytogenes. Representative confirming assays of this type aredescribed in Bergey's Manual of Systematic Bacteriology; Vol. 2, Sec.14, and particularly p. 1241, which is incorporated by reference herein.

Using the above technique, the following Listeria strains set forth inTable I have been tested, and all such pathogens are detectable in thesystem.

                  TABLE I                                                         ______________________________________                                        Listeria spp. tested                                                          Listeria spp.                                                                            Strains       Sources                                              ______________________________________                                        L. monocytogenes                                                                         102, 103, 203 Kansas State University                              (14)                     of Veterinary Medicine                                          LM101M, LM103M,                                                                             University of Wisconsin                                         Scott A                                                                       Unknown       The Pillsbury Company                                           Unknown       Russell Research Center                                         V7, ATCC 35152,                                                                             University of Arkansas                                          LCDC 81-861,                                                                  Scott A. F 5069                                                               ATCC 1911     ATCC                                                            ATCC 43259                                                         L. ivanovii                                                                              KC 1714       University of Arkansas                               (2)                                                                                      ATCC 19919    Russell Research Center                                         KC 1714                                                            L.innocua  -/-*, +/-*,   The Pillsbury Company                                (6)        5474, 5910-1,                                                                 4616, 5618                                                         L. welshimeri                                                                            +/+*, -/+*    Same as above                                        (2)                                                                           L. seeligeri                                                                             Unknown       Same as above                                        (1)                                                                           ______________________________________                                         * = Rhamnose/Xylose sugar fermentations.                                 

The following Table II sets forth assay reaction times observed usingthe above-described technique on beef samples inoculated with varyingconcentrations of L. monocytogenes strains. In each case the first-stagereaction in the Stomacher bag involved approximately 20 hours incubationtime. and thereafter 1 ml. samples of the liquid were placed in thedescribed U-tube filled with agar and liquid. The "Pre-Enrich Time" foreach sample was the incubation until a black color was observed in thegrowth liquid above the agar (i.e. liquid 24 of FIG. 1). The "Thru-AgarTime" was the incubation time observed for the pathogen to pass throughthe agar. The "Post-Enrich Time", was the time observed until theconfirming liquid 26 was blackened in the U-tube. In all instances. thetimes listed are cumulative.

                  TABLE II                                                        ______________________________________                                        Detection of L. monocytogenes from Inoculated Meats                           After First Stage Blackening in Fraser Broth                                                     Pre-Enrich                                                                    Time (hr.)                                                                    (1st Side of                                                                            Thru-Agar                                                                             Post-Enrich                              Strains Inocula.sup.1                                                                            U-Tube)   Time (hr.)                                                                            Time (hr.)                               ______________________________________                                        LM 101M 210 CFU/ml 3-4       12-13   15-16                                             21        5-6       14-15   17-18                                             2.1       7-8       15-16   18-19                                    LM 103M 340 CFU/ml 2-3        9-10   12-13                                             34        4-5       10-11   13-14                                             3.4       5-6       12-13   15-16                                    Scott A 140 CFU/ml 3-4       14-15   17-18                                             14        4-5       15-16   18-19                                             1.4       4-5       14-15   18-19                                    ______________________________________                                         .sup.1 Assumed pathogen concentration obtained using standard dilution        techniques.                                                              

EXAMPLE 2

A series of tests were undertaken to determine the effect of oxyraseenzyme upon the growth characteristics of various L. monocytogenesstrains. In all instances the tests were directly comparative, andinvolved placing identical concentrations of the respective strains inidentical quantities of growth medium, followed by incubation at 37° C.for 50 hours. In particular, each strain was tested by placing 10⁶CFU/ml. of the strain in 100 ml. of Fraser Broth; a comparative test wasundertaken using the same concentration of pathogen in the same quantityof Fraser Broth, but with the addition of 10 units of oxyrase enzyme.FIGS. 2-4 graphically depict the results of these comparative tests, anddemonstrate significantly faster growth of the L. monocytogenes strainsin the presence of oxyrase.

EXAMPLE 3

In this test, the effects of oxyrase on the growth of S. typhimuriumsuspended in TSB (Tryptic Soy Broth) and incubated at 37° C. for 20hours. Here again, the test was directly comparative, and involvedplacement of 10⁵ CFU/ml. of S. typhimurium in 100 ml. of TSB, followedby incubation for 20 hours. A comparative sample was identicallyproduced, save for the addition of 10 units of oxyrase enzyme in theTSB. The results of this test are set forth graphically in FIG. 5.

EXAMPLE 4

This test was another direct comparative study wherein the effect ofoxyrase on E. coli 0157:H7 suspended in BHI (Brain Heart InfusionMedium). In this case, a controlled sample consisting of 10⁵ CFU/ml. ofE. coli 0157:H7 was placed in 100 ml. of BHI, followed by incubation at37° C. for 22 hours. A comparative sample was also compared which wasidentical except for the addition of 10 units of oxyrase enzyme. Thisenzyme-added sample was incubated under these same conditions as thecontrol. The results of this test are set forth in FIG. 6.

EXAMPLE 5

In order to determine the effect of oxyrase enzyme on heat injured L.monocytogene, the following experiment was undertaken. Specifically,Brain Heart Infusion Broth (BHI, pH 7.4; 200 g calf brain, 50 g beefheart, 10 g protease peptone, 2 g dextrose, 5 g NaCl. and 2.5 g disodiumphosphate per liter of distilled water) in a 10 ml. test tube waspreheated at 60° C. for 1 hour in a circulating water bath. One ml. of a10⁸ suspension of L. monocytogenes was added to the preheated BHI,shaken, and held in the 60° C. water bath for exactly 15 minutes. Thebroth was then rapidly cooled in an ice bath.

One ml. of the heat-stressed cells (about 10⁴) was added into 100 ml. ofBHI Broth in a Klett flask, and the turbidity of the Broth was measuredhourly using a Klett meter, during incubation at 37° C. over a period of25 hours. A comparative sample was similarly treated, except that itincluded 10 units of oxyrase enzyme. The results of this test are setforth in FIG. 7, and demonstrate that the presence of oxyrase enhancesgrowth of the injured L. monocytogenes.

EXAMPLE 6

In this test the assay of the present invention was directly comparedwith the prior art FSIS method for the isolation and identification ofL. monocytogenes. In this experiment, samples of ground beef wereinoculated with varying concentrations of the LM 103M strain, and thesesamples were subjected to the prior art assay and that of the presentinvention, the latter being described in Example 1.

Table III sets forth the results of these tests, and will be seen thatin all instances the U-tube technique of the present invention gavesignificantly faster assay results, as compared with the prior artmethod.

                  TABLE III                                                       ______________________________________                                        L. monocytogenes                                                                              1000    100     10   1    0.1                                 ______________________________________                                        FSIS Prior Art Technique                                                                      7       8       8    9    9                                   Time (hr.) to see black                                                                       31 to 33 hr.                                                                              +      24 to 36 hr.                               color in Fraser Broth after                                                                   (enrich.)          (plating)                                  24 hr. primary enrichment                                                     Total Time      55 to 69 hrs.                                                 U-Tube Technique                                                                              18      20      22   25   26                                  Time (hr.) to see black                                                                       + ca. 20 hr. in U-Tubes                                       color in Fraser Broth +                                                       Total Time      38 to 46 hrs.                                                 ______________________________________                                    

EXAMPLE 7

The assay of the invention is highly selective for the determination ofmotile facultative anaerobic pathogens such as L. monocytogenes. A largenumber of potential competitive pathogens set forth in Table IV belowhave been determined to exhibit no growth in Fraser Broth. Hence, thepresence of these pathogens in a given sample would not detract from theaccuracy, of the assay.

                  TABLE IV                                                        ______________________________________                                        Pathogens Exhibiting No Growth in Fraser Broth                                ______________________________________                                        Acetobacter aceti  Salmonella chameleon                                       Acinetobacter calcoaceticus                                                                      Salmonella enteritidis                                     Arizona hinshawii  Salmonella molade                                          Arthrobacter globiformis                                                                         Salmonella montevideo                                      Bacillus cereus    Salmonella senftenberg                                     Bacillus subtilis  Salmonella typhimurium                                     Bordatella bronchisetica                                                                         Serratia liquefaciens                                      Citrobacter freundii                                                                             Serratia rubidaea                                          Hafnia alvei       Shigella boydii                                            Micrococcus luteus Shigella dysenteriae                                       Propionibacterium sp.                                                                            Shigella flexneri                                          Proteus mirabilis  Shigella sonnei                                            Proteus morganii   Staphylococcus albus                                       Proteus strutzer   Staphylococcus aureus                                      Providencia sturartii                                                         ______________________________________                                    

In addition. the following potentially competitive pathogens (Table V),while exhibiting growth in Fraser Broth, are not motile in the modifiedOxford agar, or give a straw color in Fraser Broth. Accordingly, thesepathogens likewise do not detract from the specificity of the presentassay.

                  TABLE V                                                         ______________________________________                                        Growth in Fraser Broth and Appearance of Dark Black                           Color but No Motility Through U-Tube                                                   Enterobacter aerogenes                                                        Enterobacter aerogenes                                                        Flavobacterium capsulatum                                                     Klebsiella oxytoca                                                            Klebsiella pneumoniae                                                         Morganella morganii                                                           Proteus vulgaricus                                                            Pseudomonas aeruginosa                                                        Pseudomonas fluorescens                                                       Pseudomonas fragi                                                             Pseudomana maltophilia                                                        Serratia marcescens                                                           Streptococcus faecalis                                                        Streptococcus lactis                                                 Growth in Fraser Broth and Appearance of Straw Color                                   Enterobacter cloacae                                                 ______________________________________                                    

EXAMPLE 8

In this Example a number of other selective enrichment brothssupplemented with esculin and iron were tested and compared with FraserBroth, insofar as growth of L. monocytogenes therein is concerned. Inparticular, standard Listeria enrichment broth (LEB), LEB supplementedwith 1.0 g/l. esculin and 0.5 g/l. of ferric ammonium citrate, FraserBroth, University of Vermont Broth (UVM), and UVM supplemented with 0.5g/l. of ferric ammonium citrate were tested. In each instance 100 ml. ofa respective broth was inoculated with 10⁴ CFU/ml. of the LM101M strainof L. monocytogenes. The respective samples were then incubated at 37°C. for 24 hours. The results of these series of tests are set forth inFIG. 8, where it will be seen that other growth media provide excellentgrowth for the pathogen, and could therefore be used in the invention.

EXAMPLE 9

In this test the effectiveness of several reducing agents was measured.In each case 10³ CFU/ml. of LM 101M L. monocytogenes was inoculated intorespective 100 ml. portions of BHI Broth. 0.05% by volume portions ofreducing agents were then added to the inoculated broth samples, alongwith oxyrase enzyme and a control (i.e., no enzyme or reducing agent).The broth samples were then incubated for eight hours at 37° C., andbacterial counts were taken. The results (Table VI) showed that thereducing agents were effective, but the oxyrase enzyme was stillsuperior.

                  TABLE VI                                                        ______________________________________                                        Effect of Reducing Agents on Growth of L. monocytogenes                       LM 101M in BHI Broth after 8 hour incubation at 37° C.                 Reducing Agent                                                                            Number of Cells                                                                             Reported E'.sub.h Value                             ______________________________________                                        L-cysteine.HCl                                                                            1.9 × 10.sup.6                                                                        -210 mV                                             Titanium III citrate                                                                      1.8 × 10.sup.6                                                                        -480 mV                                             Oxyrase     2.9 × 10.sup.6                                                                        -200 to -300 mV                                     Control     1.4 × 10.sup.6                                                                        --                                                  ______________________________________                                    

We claim:
 1. In a method of incubating a facultative anaerobicmicroorganism in order to enhance the growth rate thereof, including thesteps of placing the microorganism in a growth medium which supportsgrowth thereof, and causing said microorganism to grow in said growthmedium, the improvement which comprises the step of supplementing thegrowth medium with an effective, growth rate-enhancing amount of freeoxyrase enzyme in the growth medium.
 2. The method of claim 1, includingthe step of incubating said microorganism at a temperature of 37° C. 3.The method of claim 1, said growth medium being sterile.